Catenulin and its production



United States Patent O CATENULIN AND ITS PRODUCTION Jacob W. Davisson, Floral Park, and Alexander C. Finlay,

Flushing, N.Y., assignors to Chas. Pfizer & Co., Inc.,

Brooklyn, N.Y., a corporation of Delaware No Drawing. Application March 11, 1954 Serial No. 415,688

9 Claims. c1. 167-65) from the known species of Streptomyces listed in the accepted classification of Bergeys Manual of Determina-v tive Bacteriology (6th edition), pages 928-980. The cultural characteristics of this species, based on isolate No. 5541-6A of Chas. Pfizer & Co., Inc., are given below in tabular form. (The colors, where R is written, are those of Ridgway, Color Standards and Nomenclature.) Readings were determined on six tubes or plates. The specific name chosen for this newly isolated microorganism indicates the short chains of spores which it produces, one of its distinctive characteristics. A living culture of the organism has been deposited with the American Type Culture Collection at Washington, DC, and has been added to its permanent collection of microorganisms as ATCC 12,476.

2,895,876 Patentedv July 21,

It is to be understood that the production of catenulin is not limited to this particularorganism or to organisms fully answering the above description, which is given merely for illustrative purposes. Those organisms are especially contemplated which are mutants produced from the described bacterium by mutating agents such as X- radiation, ultravioletradiation, nitrogen mustards and so forth.

In order to measure the potency of various solutions or solid preparations of catenulin, an essay procedure is used in which is determined. the turbidity of standard cultures of a Staphylococcus aureus strain grown in a standard test medium in the presence of varying amounts of the given sample of catenulin. An arbitrary standard has been ohosen,.based on a sample of catenulin which assayed 3200 CDU/mg. in the E. coli dilution test described below. A value of 1000 catenulin units per milligram has been assigned to this preparation. The test medium is the Baltimore Biological Laboratory assay broth, prepared according to the Food and Drug Administration formula for streptomycin assay broth, and the method used is that of J. R. McMahan (J. Biol. Chem, vol. 153, pages 249-258, April 1944). v I

Caienulin possesses activity against a widevarietyof organisms. However, its activity against the usual gram positive and gram negative microorganisms is somewhat limited as compared to its activity against the mycobac- V teria. The following table shows the spectrum of catenulin against a group of gram positive and gram negative bacteria, as well as certain molds. These tests were conducted by streaking standard cultures of the microorganisms on agar plates containing varying known concentrations of a preparation of catenulin. The levels tested werel00, 50, 25, 10, 5 and '1 micrograms of catenulin per milliliter of agar medium. In the table below is given the minimum concentration within these rangesjwhich completely inhibited the growth of the streaked microorganism culture during 24 hours incubation at 37 C. The preparation of catenulin used in this series of tests had an activity of 6400 E. coli dilu- Streptomyces catenulae (ATCC 12,476)

' Color Medium Amount of Soluble Pigment Remarks 'Growth Aerial Mycelium and Spores Glucose-asparagine agar.--. Moderate to good. Moderate sporulation near Mouse Faint yellowish... Colony slightly elevated in center; 7 Gray (R); white aerial mycelium surface concentrically and radiately edge. wrinkled; sporulated surface rough and granular; smooth edge; reverse dark brown or dark greenish brown; spores in very short clustered chains; clusters very dense; a few fairly tight spirals; spores oval to short cylindrical, 1.0 x 1.32. Color of sporulated colonies on dilution plates between Dark Olive Gray (R) and Hair Brown (R); colonies alike. Gelatin plates, 21 0. Moderate spread- Waxy, near Cartridge Butt (R); some None Poor liquefaction.

ing. white aerial mycelium. Glucose agar Good Good sporulation, near Pale Drab- Yellow.. Reverse yellowish brown.

- Gray (R); waxy edge Olive-Bud (R);

some white aerial mycelium. Nutrient agar Moderate Abundant white aerial mycelium; Pale Yellow Reverse pale yellow.

slight sporulation, a very pale gray. Potato Plugs Good Good sporulation from Pale Olive (R), Dark greenish, oc- Greenish vegetative mycelium.

, Smoke Gray (R), and Hair Brown casionally no i (R). soluble pig- I ment. h Calcium malate agar Poor 'Good sporulation, near Pallid Mouse None Reverse not visible; calcium malate' Gray (R); some white aerial mydigested. e m. Starch plates --do Waxy, light brownish orange; cor .---.do Reverse light brownish orange; good sporulation near Deep Mouse ray hydrolysis of starch.

Synthetic agar Very poor spread- Sporulation light gray .-do Transparent, deep white subsurface ing. growth; reverse transparent, white. Cellulose strips- Very poor growth. Emersons agar..-- Moderate Good sporulation, near Olive-Gray Medium brown--. Reverse very dark brown.

(R); white aerial mycelium. Dextrose nitrate broth- Nitrate not reduced.

tiori units per milligram of dry solid, i.e. one milligram of the preparation diluted to 6400 milliliters just inhibited a standard culture of Escherichia coli grown in a-suitablenutrient medium for 18 hours at 37 C.

protein supplement (A.P.S.) to provide a positive control, i.e. a nutritionally balanced feedstulf supplying all of the normal requirements of the chicks. The basal ration so employed had the following composition:

TABLE I Yellow corn meal perc 52.2 1 7 Concentration of catenulin soybean on meal do 22 Microorganism: preparation, meg/ml. Middlings do ,A. ae'rogenes 1 Alfalfa leaf meal do 3 -A. aerogenes Y 1 50 Fish meal dn 4 coli 25 10 Meat scraps do 4 Proteus sp. 50 CaCO 1.8 B, btili 10 Dicalcium phosphate ....do 1 Micracoccus pyogenes var. aureus 10 Salt o...... 0.5 Str. faecalis 25 Viadex do 0.13 t Brucella bronchiseptica MnSO gm 100 1b-- 10 Sal. parazyphi B 50 Choline chloride 25% do 312 Candida albicans Thiamin do 90 .saz. typhosa 25 Riboflavin do 160 K, pneumoniae 25 Calcium pantothenate do 545 i I Sal, paratyphi A 50 Niacin 800 Ps. aeruginosa Pyridoxine do 160 Tricophym" menfagmlhytes (2) The results of these tests are summarized in the fol- Resistant: to chlortetracy cline (Aureomycin) and chlorlowing table. For the purposes of comparison, some of i fgf g $37$f the groups were fed rations supplemented with oxytetracycline hydrochloride, a hrghly eifectrve growth pro- The activity of catenulin has also been measured against moting antibiotic sold under the registered trademark a group of mycobacteria in the same manner as de- Terramycin.

TABLE 111 Average Body Weight-weeks Feed Group Supplement to Diet Supple- Net Growth Efflmenta- Gain Index ciency tion, 0 1 2 3 4 -l Grams Grams Grams Grams Grams Positive Control (P)- 87 154 259 339 294 100 2.15 P+catenulin 2 42 88 164 273 368 32s 103 2.09 4 do 5 4s 92 174 282 391 346 115 2.02 4 do 10 45 92 164 268 388 343 114 2.08 5 P-i-oxytetracycline H01..... 2 42 93 111 281 388 346 114 1.91 a do 5 42 9a 168 276 320 338 112 2.02 7 do 10 42 84 158 260 364 322 107 2.12

scribed above and, using the same preparation. The In the above table growth index is a measure of growth compound shows an unexpectedly high order of activity response (in terms of weight) to the antibiotic suppleagainst this group of microorganisms (Table II). The 4 mentation in relation to the basal ration or positive preparation here used was prepared in accordance with control without such supplementation, the higher the Example I below. index'the greater the response. The positive control is arbitrarily assigned a value of 100 to provide a basis for TABLE II cncentmfionof comparison. Feed efiiciency is the ratio of the weight of Mycobacterium: catenulin, meg/m1, feed required per unit weight of growth increase, the Mycobacterz'um ranae 5 lower the ratio the higher the efficiency of the particular Mycobacterium phlei 3 .feed. Mycabacterium smegmatis 5 From theabove tests it is apparent that catenulin sup- Mycobacterium species No. 607 5 ported increases in the growth of chicks ranging upward Comparable activity is demonstrated against huto 15% over the controls at the levels of catenulin sup- 1 man strains of Mycobacterium tuberculosis. plementation employed. It is further apparent that catenulin supports growth increases in chicks comparable The marked specificity of catenulin against mycobacto the increases obtainable with oxytetracycline. Additeria, as compared to its activity against other types of tional tests with catenulin have confirmed these results. microorganisms, is a rather unique property of the sub- Catenulin has been distinguished from known antibistance and further illustrates its marked difierence from otics by its antimicrobial spectrum and by a number of most known antibiotics, such as penicillin, streptomycin, other properties. For instance, its behavior when chrooxytetracycline (Terramycin), chlortetracycline (Aurematographed on paper, using various solvent systems, is omycin), chloramphenicol (Chloromycetin), streptodistinct from the various compounds heretofore menthricin, etc. While utility of the antibiotic in human tioned. Further, it is not appreciably extracted .or flushed therapy has not as yet been demonstrated, it is parfrom its dilute aqueous broth solutions into the usual ticularly useful in promoting the growth of animals. solvents at acid, neutral, or basic pHs, as are penicillin A series of tests were conducted with catenulin to and the like materials. It also possesses a quite high dedetermine its effects on the growth of chicks. In these gree of stability. It can be maintained at a lowered tests the chicks were divided into groups and fed a 7 or raised pH for appreciable time without undue loss, basal ration supplemented with various proportions of although heating for extended periods in an acid medium catenulin, in the form of the sulfate, over a period of 7 tends to decrease its activity. Our new biologically acfour weeks, during which the average weight, growth retive substance contains the elements carbon, hydrogen, spouse and feed efliciency were recorded for each group. oxygen andnitrogen, but no sulfur, phosphorus, or halo- The basal ration was supplemented with 8% animal 'lli gen.

Catenulinhas a low order of toxicity. Thus, a sample of thematerial having a potency of 6400 E. coli dilution units per milligram (CDU/mg.) was found, on intravenous injection into mice, to have an LD of 4.5 milligrams pergmouse. 'Ihe LD for the same animal, also using intravenous administration, was 6.0 milligrams.

This invention also embraces processes for growing the hitherto unknownspecies of microorganism, Streptomyces catenulae. Such processes involve cultivating the ibacterium in a suitable nutritive medium, preferably at about 22 to 32 C. under aerobic conditions and with agitation. The standard culture media contain a source of carbohydrate, such as sugars, starch and glycerol; a source of organic nitrogen, such as bean meals and particularly soybean meal, wheat gluten, cotton seed meal, lactalbumin, and enzymatic digests of proteins; and a growth-promoting substance, such as distillers solubles and yeast extracts. Mineral salts like sodium chloride, sodium nitrate, and potassium phosphate, a bufiering agent like calcium carbonate and a vegetable or mineral oil are usually also incorporated therein. After growth for a suitable time until an appreciable antibiotic potency has been imparted to the medium, generally a period of about one to five days, the mycelium which is formed is usually separated from the broth containing the elaborated catenulin, and the latter is then recovered by various procedures.

It is anticipated that there will be certain moieties within the catenulin chemical structure, which will be useful as precursors for the preparation of the substance by fermentation.

Inoculum for the commercial preparationof catenulin may be obtained from growth inslants or Roux bottles inoculated with a strain of Streptomyces catenulae. Solid media suitable for this initial growth of the microorganism are e.g., beef lactose or Emersons agar. Such growth may then be used to inoculate either shaken flasks or inoculum tanks for submerged aerobic cultivation. Alternatively, inoculum tanks may be later inoculated from shaken flasks after the latter have been cultivated for a suitable period. The growth in shaken flasks generally reaches its maximum in two to four days. After a suitable period of incubation, the contents of the inoculum tanks may be added to larger fermenters under aseptic conditions and cultivation in the new medium continued for one to five days. In the inoculum tanks and in the large scale fermenters, agitation is preferably continued throughout the fermentation by blowing in sterile air through spargers at the rate of about /2 to 2 volumes of free air per volume of broth per minute and by stirring at speeds of 100 to 2000 rpm. If foaming is encountered during the growth, it may be controlled by the addition of suitable foam-controller such as vegetable oil, mineral oil, a silicone, octanol or the like. When grown in this manner, Streptomyces catenulae furnishes broths containing from about 200 to about 5000 CDU/ ml. of the catenulin.

The catenulin may be recovered by a variety of meth,-"

ods from fermentation brothsproduced by the methods described above. The solid mycelial growth should first be removed from the broth by filtration. It is preferable to acidi fy the mixture before this clarification, in order to achieve maximum separation of the catenulin from the mycelia, and a filteraid may also be employed to assure a broth of high clarity. The biologically active substance may then be removed from the filtrate by adsorption at about a neutral pH on solid adsorbents like carbon or cation-exchange resins (e.g. of the carboxylic or sulfonic acid types). Various organic solvents such as methanol, ethanol or acetone, either dry or containing water, and preferably containing some acid, are utilized to elute the substance from carbon, while dilute acids like hydrochloric, sulfuric or acetic serve in the elution of resinous adsorbates. The eluates are finally neutralized and dried to recover products of elevated catenulin activity. Such products may be used directly in therapy or purified still further by conventional means.

Another very useful method of isolating and recover ing catenulin from its fermentation broths is by precipitation with various arylazosulfonic acid dyes. These materials readily form salts with the substance which have a low degree ofsolubility. The precipitates are generally prepared with the broth at a pH of about 5 to 8, and the salt products may be filtered from the exhausted broth. If difficulty is encountered with the filtration, a suitable filteraid may be utilized. The solid dye salts of catenulin may be used as such or further treated toregenerate a purified form of the substance. Among the particularly useful dyes are Eriochrome Violet, Orange II, Helianthine, Naphthol Blue-Black, Polar Yellow, Fast Red. These have respectively the Color Index numbers 169, 151, 150, 246, 642 and 176. Of these the first is especially satisfactory in commercial operations; Recovery of the catenulin from such dye salts is accomplished in various ways. One preferred method is to treat a solvent solution thereof with an amine salt of an acid, which salt is soluble in the chosen solvent but .in turn forms a solventinsoluble salt with the catenulin. Triethylamine sulfate in methanol is a most suitable combination. A metallic salt may he used instead of an amine salt. Thus, if an arylazosulfonic acid salt of catenulin is treated in a solvent with barium chloride, the barium salt of the dye will separate and a solution of the catenulin hydrochloride is obtained. It is obvious that metallic salts other than barium chloride may be used, the only limitations being that the dye salt of the metal used be insoluble in the chosen solvent and the acid salt of the catenulin formed during the metathetical reaction be soluble in that solvent. By removal of the solvent from the filtered solution, a dry biologically active product is finally obtained. A further method for converting arylazosulfonic acid salts of catenulin is by treatment with a suitable dilute solution of a strong acid, such as sulfuric acid or hydrochloric acid. This results in the formation of the free arylazosulfonic acid, which has a limited solubility in water and lower alcohols. The dye may then be removed by filtration and any traces thereof remaining in the solution adsorbed therefrom by activated carbon or the like. The resulting filtrate containing an acid salt of catenulin in solution may be evaporated to yielda dry preparation of the substance. Rather than filtering the arylazosulfonic acid, it may be extracted with a solvent such as butyl alcohol or amyl alcohol.

These various purification methods may be repeated or may be used in combination with one another to obtain products of increased purity. In addition, the antibiotic may be separated from various inorganic salt impurities by selective extraction of a dry sample with a suitable solvent. Materials prepared by the above procedures have been shown by paper chromatography, using various solvent systems, to contain only the one compound, catenulin.

A particularly useful procedure whereby catenulin may be highly purified involves the formation of the helianthate of this compound which salt has been obtained in crystalline form. Thus, if a sample of a simple catenulin salt, such as the sulfate"orhydrochloride, assaying about 1500 catenulin units per mg. or higher, is treated in aqueous solution with soluble helianthate such as an amine salt of helianthic acid (pyridine helianthate, triethylamine helianthate, and so forth), there is obtained the crystalline helianthate. This compoundmay be recrystallized from a solvent such as methanol containing a major proportion of water. Other lower alcohols or ketones may also be used. Crystalline catenulin helianthate does not melt below 300 C., butit does darken slightly attemperatures above 240 C. It consists of fine red-brown needles when-prepared as described above. This crystalline salt has an assay of about 1200 catenulin units per mg. Inaddition to the helianthate a crystalline 'reineckate has been obtained by treating the catenulin in water with ammonium reineckate.

From the helianthate it is possible to regenerate highly purified preparations of simple salts of catenulin such as the-hydrochloride or sulfate. One method of doing this is to dissolve the crystalline salt in methanol and then treat the solution with concentrated hydrochloric acid. Crystalline helianthic acid separates and may be removed by filtration. Small amounts of dye left in the solution may be removed by a decolorizing agent and catenulin hydrochloride may then be recovered by a method such as precipitation from a solvent in which it has low solubility, e.g. acetone.

The p(p'-hydroxyphenylazo)-benzene sulfonic acid salt of catenulin has also been crystallized, and the simple sulfate salt regenerated therefrom by treatment in methanol with triethylamine sulfate and drying (e.g. in vacuum at 100 C.). The sulfonic acid compound analyzes about 50.24% by weight of carbon, 5.29% hydrogen, 10.01% nitrogen and 8.00% sulfate. It has an optical rotation of lul =+33 (1% in methanol). The sulfate analyzes about 31.53% by weight carbon, 6.0% hydrogen, 7.92% nitrogen and 28.11% sulfate; its optical rotation is Example I A medium containing water and the following proportion of ingredients by weight was prepared:

Percent Cerelose L Sodium chlor de 05 Distillers solu l s 05 Enzymatic hydrolysate of casein 0.5

I After this solution had been prepared it was adjusted to pH 7 with potassium hydroxide and 0.1% of calcium carbonate was added. A small amount of a vegetable oil as an anti-foaming agent was added. The liquid was dispensed to Fernbach flasks, 500 milliliters in each. The flasks were plugged with cotton and were sterilized at 25 pounds of steam pressure for 45 minutes. The pH of the mixture had decreased to 6.5 This nutrient medium was inoculated from a slant of Streptomyces catenulae ATCC 12,476 which had been grown on beef lactose agar. The flasks were shaken for two days at 28 C. on a rotary shaker. One liter of this inoculum was then added under sterile conditions to 20 gallons of the same type of nutrient medium, which had previously been sterilized and cooled in an inoculum tank equipped for submerged fermentation. This inoculated mixture was stirred and aerated at the rate of about one volume of air per, volume of rri ediur'nper minute for one day, while the temperature was 'maintained at 27 C.

The contents of the inoculum tank were forced under aseptic conditions into 150 gallons of the same type medium, which had been sterilized and cooled. This mixture was subjected to aeration and rapid stirring for a period of 43 hours at a temperature of about 27 to 28 C. After 41 hours of growth the mixture had a catenulin potency of 320 CDU/ml. The broth was adjusted to pH 5.6 with dilute: sulfuric acid, pounds of a diatomaceous earth filtereaid were added, and the material was thoroughly stirred and filtered. To the clear filtrate were added 900 grains of Eriochrome Violet. The mixture was stirred for half an hour and filtered with a filteraid. The filtercake was washed several times with water, to remove adhering exhausted broth, and then dried. Q

One-half of this filtercake was'added to 12 liters of methanol. A solution of 2500 milliliters of 50% triethylamine sulfate in methanol was slowly added to the mixture. After stirring for one hour, the catenulin sulfate salt, mixed with filteraid, was removed by filtration and washed by slurrying three times with six liters of methanol and one time with six liters of acetone. Practically all of the dye was removed by this treatment. The filtercake obtained after the last wash was sucked free of solvent and then suspended in 2500 milliliters of water. It was filtered to remove filteraid and washed with a small volume of water. The resulting clear, aqueous solution was dried under vacuum from the frozen state The product so obtained was dissolved in a small volume of water (600 ml.). A small amount of insoluble material, apparently inorganic salts, was removed by filtration and the filtrate was again dried from the frozen state. The product weighed 6.0 grams and assayed 6400 CDU/mg. This product was particularly active against mycobacteria and in promoting the growth of chicks; it possessed a relatively low degree of toxicity.

Example II A sample of 5.0 grams of catenulin sulfate obtained as in the example above and assaying 1675 catenulin units per mg. was dissolved in 10 m1. of water. Five ml. of a saturated aqueous ammonium oxalate solution was added to remove calcium. The precipitate was centrifuged and the supernatant liquid was removed. This solution was treated with ml. of an aqueous solution containing 10 grams of helianthic acid as its pyridine salt. The slow addition of this reagent caused the formation of crystalline catenulin helianthate in the form of fine red-brown needles. These were separated by centrifugation and were washed by suspension in hot water. After the suspension had been filtered the product was dried. It weighed 14 grams and assayed 1200 catenulin units per mg. A sample recrystallized twice from 33% methanol did not melt up to a temperature of 300 C., but slight darkening of the sample occurred at 240 C.

Example Ill Five grams of crystalline catenulin helianthate pre pared as in the example above was dissolved in 50 ml. of methanol. To the solution was slowly added 2 ml. of concentrated hydrochloric acid. The solution was stirred during the addition of the addition of the acid. The crystalline helianthic acid that separated was filtered and the purple solution was passed through a bed of a decolorizing carbon (Darco G60). The clear, practically colorless solution was then added slowly to 5 volumes of acetone with stirring. The precipitated catenulin hydrochloride was filtered and dried. It weighed 1.25 grams and assayed 3740 catenulin units per mg. As many apparently widely different embodiments of this invention may bemade without departing from the spirit and scope hereof, it is to be understood that the above inventionis not limited except as defined in the appended claims.

What is claimed is: I

1. A process for producing oatenulin, which comprises cultivating Streptomyces catenulae under submerged aerobic conditions in an aqueous nutritive medium containing a growth-promoting substance, carbohydrate and organic nitrogen, until substantial antibacterial activity is imparted to said medium. Y

2, A process for producing catenulin, which comprises growing Streptomyces catenulae for between about one and five days under submerged aerobic conditions with agitation in an aqueous solution maintained at a temperature of substantially from 22 to 32 C. and containing a growth-promoting substance, carbohydrate and organic 9 nitrogen, and then recovering from the broth the eatenulin thus elaborated.

3. The process of claim 2 wherein the recovery of the catenulin includes the operations of clarifying the broth, adsorbing the catenulin from the clarified broth with a solid absorbent, and eluting the absorbate.

4. The process of claim 2 wherein the catenulin is recovered as a salt by precipitation from the broth with an arylazosulfonic acid dye selected from the class consisting of Eriochrome Violet, Orange II, helianthine, naphthol blue-black, Polar Yellow and Fast Red.

5. A new biologically active substance identified as catenulin, which is prepared by the process of claim 1,

7. A hydrochloride of the substance claimed in claim 6. v

8. A sulfate of the substance claimed in claim 6. 9. A helianthate of the substance claimed in claim 6.

References Cited in the file of this patent UNITED STATES PATENTS 2,462,175 Folkers Feb. 22, 1949 10 p Duggar Sept. 13, 1949 Sobin July 18, 1950 OTHER REFERENCES Annals of the N.Y. Acad. of Sci., vol. 60, art. 1, pp. 1-182, 5, 24, 79-80 and 150, 1954.

Bergeys Manual of Determinative Bacteriology, 6th ed., pp. 47-48, 1948, by Williams and Wilkins Co.

Gore et al.: Art. in Annals of the N.Y. Acad. of Sci., vol. 51, art. 5, page 924.

Waksman: The Actinomycetes, pp. 116, 117 and 121, pub. 1950, Chronica Botanica Co., Waltham, Mass.

Waksman et al.: Neomycin, Science, March 25, 1949, vol. 109, No. 2830, pp. 305-307, Ql-S-34.

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Actinomycetes and Their Antibiotics, by Waksman et al., pp. 174, 199, 200. Publ. by Williams and Wilkins Co., Baltimore, Md.

Davisson et al.: Antibiotics and Chemotherapy, pp. 460-461, September 1952. 

1. A PROCESS FOR PRODUCING CATENULIN, WHICH COMPRISES CULTIVATING STREPTOMYCES CATENULAE UNDER SUBMERGED AEROBIC CONDITIONS IN AN AQUEOUS NUTRITIVE MEDIUM CONTAINING A GROWTH-PROMOTING SUBSTANCE, CARBOHYDRATE AND ORGANIC NITROGEN, UNTIL SUBSTANTIAL ANTIBACTERIAL ACTIVITY IS IMPARTED TO SAID MEDIUM. 